Human RNase H and compositions and uses thereof

ABSTRACT

The present invention provides polynucleotides and polypeptides encoded thereby of human Type 2 RNase H. Methods of using these polynucleotides and polypeptides in enhancing antisense oligonucleotide therapies are also provided.

This application is a continuation of U.S. Ser. No. 09/343,809, filedJun. 30, 1999, now abandoned which is a continuation of U.S. Ser. No.09/203,716, filed Dec. 2, 1998 and issued as U.S. Pat. No. 6,001,653 onDec. 14, 1999 which claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 60/067,458, filed Dec. 4, 1997.

FIELD OF THE INVENTION

The present invention relates to a human Type 2 RNase H which has nowbeen cloned, expressed and purified to electrophoretic homogeneity andhuman RNase H and compositions and uses thereof.

BACKGROUND OF THE INVENTION

RNase H hydrolyzes RNA in RNA-DNA hybrids. This enzyme was firstidentified in calf thymus but has subsequently been described in avariety of organisms (Stein, H. and Hausen, P., Science, 1969, 166,393-395; Hausen, P. and Stein, H.,Eur. J. Biochem., 1970, 14, 278-283).RNase H activity appears to be ubiquitous in eukaryotes and bacteria(Itaya, M. and Kondo K. Nucleic Acids Res., 1991, 19, 4443-4449; Itayaet al., Mol. Gen. Genet., 1991 227, 438-445; Kanaya, S., and Itaya, M.,J. Biol. Chem., 1992, 267, 10184-10192; Busen, W., J. Biol. Chem., 1980,255, 9434-9443; Rong, Y. W. and Carl, P. L., 1990, Biochemistry 29,383-389; Eder et al., Biochimie, 1993 75, 123-126). Although RNase Hsconstitute a family of proteins of varying molecular weight, nucleolyticactivity and substrate requirements appear to be similar for the variousisotypes. For example, all RNase Hs studied to date function asendonucleases, exhibiting limited sequence specificity and requiringdivalent cations (e.g., Mg²⁺, Mn²⁺) to produce cleavage products with 5′phosphate and 3′ hydroxyl termini (Crouch, R. J., and Dirksen, M. L.,Nuclease, Linn, S, M., & Roberts, R. J., Eds., Cold Spring HarborLaboratory Press, Plainview, N.Y. 1982, 211-241).

In addition to playing a natural role in DNA replication, RNase H hasalso been shown to be capable of cleaving the RNA component of certainoligonucleotide-RNA duplexes. While many mechanisms have been proposedfor oligonucleotide mediated destabilization of target RNAs, the primarymechanism by which antisense oligonucleotides are believed to cause areduction in target RNA levels is through this RNase H action. Monia etal., J. Biol. Chem., 1993, 266:13, 14514-14522. In vitro assays havedemonstrated that oligonucleotides that are not substrates for RNase Hcan inhibit protein translation (Blake et al., Biochemistry, 1985, 24,6139-4145) and that oligonucleotides inhibit protein translation inrabbit reticulocyte extracts that exhibit low RNase H activity. However,more efficient inhibition was found in systems that supported RNase Hactivity (Walder, R. Y. and Walder, J. A., Proc. Nat'l Acad. Sci. USA,1988, 85, 5011-5015; Gagnor et al., Nucleic Acid Res., 1987, 15,10419-10436; Cazenave et al., Nucleic Acid Res., 1989, 17, 4255-4273;and Dash et al., Proc. Nat'l Acad. Sci. USA, 1987, 84, 7896-7900.

Oligonucleotides commonly described as “antisense oligonucleotides”comprise nucleotide sequences sufficient in identity and number toeffect specific hybridization with a particular nucleic acid. Thisnucleic acid or the protein(s) it encodes is generally referred to asthe “target.” Oligonucleotides are generally designed to bind eitherdirectly to mRNA transcribed from, or to a selected DNA portion of, apreselected gene target, thereby modulating the amount of proteintranslated from the mRNA or the amount of mRNA transcribed from thegene, respectively. Antisense oligonucleotides may be used as researchtools, diagnostic aids, and therapeutic agents.

“Targeting” an oligonucleotide to the associated nucleic acid, in thecontext of this invention, also refers to a multistep process whichusually begins with the identification of the nucleic acid sequencewhose function is to be modulated. This may be, for example, a cellulargene (or mRNA transcribed from the gene) whose expression is associatedwith a particular disorder or disease state, or a foreign nucleic acidfrom an infectious agent. The targeting process also includesdetermination of a site or sites within this gene for theoligonucleotide interaction to occur such that the desired effect,either detection or modulation of expression of the protein, willresult.

RNase HI from E.coli is the best-characterized member of the RNase Hfamily. The 3-dimensional structure of E.coli RNase HI has beendetermined by x-ray crystallography, and the key amino acids involved inbinding and catalysis have been identified by site-directed mutagenesis(Nakamura et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 11535-11539;Katayanagi et al., Nature, 1990, 347, 306-309; Yang et al., Science,1990, 249, 1398-1405; Kanaya et al., J. Biol. Chem., 1991, 266,11621-11627). The enzyme has two distinct structural domains. The majordomain consists of four α helices and one large β sheet composed ofthree antiparallel β strands. The Mg²⁺ binding site is located on the βsheet and consists of three amino acids, Asp-10, Glu-48, and Gly-11(Katayanagi et al., Proteins: Struct., Funct., Genet., 1993, 17,337-346). This structural motif of the Mg²⁺ binding site surrounded by βstrands is similar to that in DNase I (Suck, D., and Oefner, C., Nature,1986, 321, 620-625). The minor domain is believed to constitute thepredominant binding region of the enzyme and is composed of an a helixterminating with a loop. The loop region is composed of a cluster ofpositively charged amino acids that are believed to bindelectrostatistically to the minor groove of the DNA/RNA heteroduplexsubstrate. Although the conformation of the RNA/DNA substrate can vary,from A-form to B-form depending on the sequence composition, in generalRNA/DNA heteroduplexes adopt an A-like geometry (Pardi et al.,Biochemistry, 1981, 20, 3986-3996; Hall, K. B., and Mclaughlin, L. W.,Biochemistry, 1991, 30, 10606-10613; Lane et al., Eur. J. Biochem.,1993, 215, 297-306). The entire binding interaction appears to comprisea single helical turn of the substrate duplex. Recently the bindingcharacteristics, substrate requirements, cleavage products and effectsof various chemical modifications of the substrates on the kineticcharacteristics of E.coli RNase HI have been studied in more detail(Crooke, S. T. et al., Biochem. J., 1995, 312, 599-608; Lima, W. F. andCrooke, S. T., Biochemistry, 1997, 36, 390-398; Lima, W. F. et al., J.Biol. Chem., 1997, 272, 18191-18199; Tidd, D. M. and Worenius, H. M.,Br. J. Cancer, 1989, 60, 343; Tidd, D. M. et al., Anti-Cancer Drug Des.,1988, 3, 117.

In addition to RNase HI, a second E.coli RNase H, RNase HII has beencloned and characterized (Itaya, M., Proc. Natl. Acad. Sci. USA, 1990,87, 8587-8591). It is comprised of 213 amino acids while RNase HI is 155amino acids long. E. coli RNase HIM displays only 17% homology withE.coli RNase HI. An RNase H cloned from S. typhimurium differed fromE.coli RNase HI in only 11 positions and was 155 amino acids in length(Itaya, M. and Kondo K., Nucleic Acids Res., 1991, 19, 4443-4449; Itayaet al., Mol. Gen. Genet., 1991, 227, 438-445). An enzyme cloned from S.cerevisae was 30% homologous to E.coli RNase HI (Itaya, M. and Kondo K.,Nucleic Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet.,1991, 227, 438-445). Thus, to date, no enzyme cloned from a speciesother than E. coli has displayed substantial homology to E.coli RNase HII.

Proteins that display RNase H activity have also been cloned andpurified from a number of viruses, other bacteria and yeast(Wintersberger, U. Pharmac. Ther., 1990, 48, 259-280). In many cases,proteins with RNase H activity appear to be fusion proteins in whichRNase H is fused to the amino or carboxy end of another enzyme, often aDNA or RNA polymerase. The RNase H domain has been consistently found tobe highly homologous to E.coli RNase HI, but because the other domainsvary substantially, the molecular weights and other characteristics ofthe fusion proteins vary widely.

In higher eukaryotes two classes of RNase H have been defined based ondifferences in molecular weight, effects of divalent cations,sensitivity to sulfhydryl agents and immunological cross-reactivity(Busen et al., Eur. J. Biochem., 1977, 74, 203-208). RNase H Type 1enzymes are reported to have molecular weights in the 68-90 kDa range,be activated by either Mn²⁺ or Mg²⁺ and be insensitive to sulfhydrylagents. In contrast, RNase H Type 2 enzymes have been reported to havemolecular weights ranging from 31-45 kDa, to require Mg²⁺ to be highlysensitive to sulfhydryl agents and to be inhibited by Mn²⁺ (Busen, W.,and Hausen, P., Eur. J. Biochem., 1975, 52, 179-190; Kane, C. M.,Biochemistry, 1988, 27, 3187-3196; Busen, W., J. Biol. Chem., 1982, 257,7106-7108.).

An enzyme with Type 2 RNase H characteristics has been purified to nearhomogeneity from human placenta (Frank et al., Nucleic Acids Res., 1994,22, 5247-5254). This protein has a molecular weight of approximately 33kDa and is active in a pH range of 6.5-10, with a pH optimum of 8.5-9.The enzyme requires Mg²⁺ and is inhibited by Mn²⁺ and n-ethyl maleimide.The products of cleavage reactions have 3′ hydroxyl and 5′ phosphatetermini.

Despite the substantial information about members of the RNase familyand the cloning of a number of viral, prokaryotic and yeast genes withRNase H activity, until now, no mammalian RNase H had been cloned. Thishas hampered efforts to understand the structure of the enzyme(s), theirdistribution and the functions they may serve.

In the present invention, a cDNA of human RNase H with Type 2characteristics and the protein expressed thereby are provided.

SUMMARY OF THE INVENTION

The present invention provides polypeptides which have been identifiedas novel human Type 2 RNase H by homology between the amino acidsequence set forth in FIG. 1 and known amino acid sequences of chicken,yeast and E. coli RNase H1 as well as an EST deduced mouse RNase Hhomolog. In accordance with this aspect of the present invention, as apreferred embodiment, a sample of E. coli DH5α containing a BLUESCRIPT®plasmid containing a human cDNA nucleic acid molecule encoding a humanType 2 RNase H polypeptide deposited as ATCC Deposit No. ATCC 98536.

The present invention also provides polynucleotides that encode humanType 2 RNase H, vectors comprising nucleic acids encoding human RNase H,host cells containing such vectors, antibodies targeted to human Type 2RNase H, human Type 2 RNase H--his-tag fusion peptides, nucleic acidprobes capable of hybridizing to a nucleic acid encoding a human RNase Hpolypeptide. Pharmaceutical compositions which include a human Type 2RNase H polypeptide or a vector encoding a human Type 2 RNase Hpolypeptide are also provided. These compositions may additionallycontain an antisense oligonucleotide.

The present invention is also directed to methods of enhancing antisenseinhibition of expression of a target protein via use of human Type 2RNase H. Methods of screening for effective antisense oligonucleotidesand of producing effective antisense oligonucleotides using human Type 2RNase H are also provided.

Yet another object of the present invention is to provide methods foridentifying agents which modulate activity and/or levels of human Type 2RNase H. In accordance with this aspect, the polynucleotides andpolypeptides of the present invention are useful for research,biological and clinical purposes. For example, the polynucleotides andpolypeptides are useful in defining the interaction of human Type 2RNase H and antisense oligonucleotides and identifying means forenhancing this interaction so that antisense oligonucleotides are moreeffective at inhibiting their target mRNA.

Yet another object of the present invention is to provide a method ofprognosticating efficacy of antisense therapy of a selected diseasewhich comprises measuring the level or activity of human RNase H in atarget cell of the antisense therapy. Similarly, oligonucleotides can bescreened to identify those oligonucleotides which are effectiveantisense agents by measuring binding of the oligonucleotide to thehuman Type 2 RNase H.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a human Type 2 RNase H primary sequence (286 aminoacids; SEQ ID NO: 1) and sequence comparisons with chicken (293 aminoacids; SEQ ID NO: 2), yeast (348 amino acids; SEQ ID NO: 3) and E. coliRNase H1 (155 amino acids; SEQ ID NO: 4) as well as an EST deduced mouseRNase H homolog (GenBank accession no. AA389926 and AA518920; SEQ ID NO:5). Boldface type indicates amino acid residues identical to human. “@”indicates the conserved amino acid residues implicated in E. coli RNaseH1 Mg²⁺ binding site and catalytic center (Asp-10, Gly-11, Glu-48 andAsp-70). “*” indicates the conserved residues implicated in E. coliRNases H1 for substrate binding.

DETAILED DESCRIPTION OF THE INVENTION

A Type 2 human RNase H has now been cloned and expressed. The enzymeencoded by this cDNA is inactive against single-stranded RNA,single-stranded DNA and double-stranded DNA. However, this enzymecleaves the RNA in an RNA/DNA duplex and cleaves the RNA in a duplexcomprised of RNA and a chimeric oligonucleotide with 2′ methoxy flanksand a 5-deoxynucleotide center gap. The rate of cleavage of the RNAduplexed with this so-called “deoxy gapmer” was significantly slowerthan observed with the full RNA/DNA duplex. These properties areconsistent with those reported for E.coli RNase H1 (Crooke et al.,Biochem. J., 1995, 312, 599-608; Lima, W. F. and Crooke, S. T.,Biochemistry, 1997, 36, 390-398). They are also consistent with theproperties of a human Type 2 RNase H protein purified from placenta, asthe molecular weight (32 kDa) is similar to that reported by Frank etal., Nucleic Acids Res., 1994, 22, 5247-5254) and the enzyme isinhibited by Mn²⁺. Accordingly, we refer to the newly cloned human RNaseH as Type 2 RNase H or human RNase H1.

Thus, in accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode human Type 2 RNase Hpolypeptides. By “polynucleotides” it is meant to include any form ofRNA or DNA such as mRNA or cDNA or genomic DNA, respectively, obtainedby cloning or produced synthetically by well known chemical techniques.DNA may be double- or single-stranded. Single-stranded DNA may comprisethe coding or sense strand or the non-coding or antisense strand.

Methods of isolating a polynucleotide of the present invention viacloning techniques are well known. For example, to obtain the cDNAcontained in ATCC Deposit No. 98536, primers based on a search of theXREF database were used. An approximately 1 Kb cDNA corresponding to thecarboxy terminal portion of the protein was cloned by 3′ RACE. Sevenpositive clones were isolated by screening a liver cDNA library withthis 1 Kb cDNA. The two longest clones were 1698 and 1168 base pairs.They share the same 5′ untranslated region and protein coding sequencebut differ in the length of the 3′ UTR. A single reading frame encodinga 286 amino acid protein (calculated mass: 32029.04 Da) was identified(FIG. 1). The proposed initiation codon is in agreement with themammalian translation initiation consensus sequence described by Kozak,M., J. Cell Biol., 1989, 108, 229-241, and is preceded by an in-framestop codon. Efforts to clone cDNA's with longer 5′ UTR's from both humanliver and lymphocyte cDNA's by 5′ RACE failed, indicating that the1698-base-pair clone was full length.

In a preferred embodiment, the polynucleotide of the present inventioncomprises the nucleic acid sequence of the cDNA contained within ATCCDeposit No. 98536. The deposit of E. coli DH5α containing a BLUESCRIPT®plasmid containing a human Type 2 RNase H cDNA was made with theAmerican Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md.20852, USA, on Sep. 4, 1997 and assigned ATCC Deposit No. 98536. Thedeposited material is a culture of E. coli DH5α containing a BLUESCRIPT®plasmid (Stratagene, La Jolla Calif.) that contains the full-lengthhuman Type 2 RNase H cDNA. The deposit has been made under the terms ofthe Budapest Treaty on the international recognition of the deposit ofmicro-organisms for the purposes of patent procedure. The culture willbe released to the public, irrevocably and without restriction to thepublic upon issuance of this patent. The sequence of the polynucleotidecontained in the deposited material and the amino acid sequence of thepolypeptide encoded thereby are controlling in the event of any conflictwith the sequences provided herein. However, as will be obvious to thoseof skill in the art upon this disclosure, due to the degeneracy of thegenetic code, polynucleotides of the present invention may compriseother nucleic acid sequences encoding the polypeptide of FIG. 1 andderivatives, variants or active fragments thereof.

Another aspect of the present invention relates to the polypeptidesencoded by the polynucleotides of the present invention. In a preferredembodiment, a polypeptide of the present invention comprises the deducedamino acid sequence of human Type 2 RNase H provided in FIG. 1 as SEQ IDNO: 1. However, by “polypeptide” it is also meant to include fragments,derivatives and analogs which retain essentially the same biologicalactivity and/or function as human Type 2 RNase H. Alternatively,polypeptides of the present invention may retain their ability to bindto an antisense-RNA duplex even though they do not function as activeRNase H enzymes in other capacities. In another embodiment, polypeptidesof the present invention may retain nuclease activity but withoutspecificity for the RNA portion of an RNA/DNA duplex. Polypeptides ofthe present invention include recombinant polypeptides, isolated naturalpolypeptides and synthetic polypeptides, and fragments thereof whichretain one or more of the activities described above.

In a preferred embodiment, the polypeptide is prepared recombinantly,most preferably from the culture of E. coli of ATCC Deposit No. 98536.Recombinant human RNase H fused to histidine codons (his-tag; in thepresent embodiment six histidine codons were used) expressed in E.colican be conveniently purified to electrophoretic homogeneity bychromatography with Ni-NTA followed by C4 reverse phase HPLC. Thepurified recombinant polypeptide of SEQ ID NO: 1 is highly homologous toE.coli RNase H, displaying nearly 34% amino acid identity with E.coliRNase H1. FIG. 1 compares the protein sequences deduced from human RNaseH cDNA (SEQ ID NO: 1) with those of chicken (SEQ ID NO: 2), yeast (SEQID NO: 3) and E.coli RNase HI (Gene Bank accession no. 1786408; SEQ IDNO: 4), as well as an EST deduced mouse RNase H homolog (Gene Bankaccession no. AA389926 and AA518920; SEQ ID NO: 5). The deduced aminoacid sequence of human RNase H (SEQ ID NO: 1) displays strong homologywith yeast (21.8% amino acid identity), chicken (59%), E.coli RNase HI(33.6%) and the mouse EST homolog (84.3%). They are all small proteins(<40 KDa) and their estimated pIs are all 8.7 and greater. Further, theamino acid residues in E.coli RNase HI thought to be involved in theMg²⁺ binding site, catalytic center and substrate binding region arecompletely conserved in the cloned human RNase H sequence (FIG. 1).

The human Type 2 RNase H of SEQ ID NO: 1 is expressed ubiquitously.Northern blot analysis demonstrated that the transcript was abundant inall tissues and cell lines except the MCR-5 line. Northern blot analysisof total RNA from human cell lines and Poly A containing RNA from humantissues using the 1.7 kb full length probe or a 332-nucleotide probethat contained the 5′ UTR and coding region of human RNase H cDNArevealed two strongly positive bands with approximately 1.2 and 5.5 kbin length and two less intense bands approximately 1.7 and 4.0 kb inlength in most cell lines and tissues. Analysis with the 332-nucleotideprobe showed that the 5.5 kb band contained the 5′ UTR and a portion ofthe coding region, which suggests that this band represents apre-processed or partially processed transcript, or possibly analternatively spliced transcript. Intermediate sized bands may representprocessing intermediates. The 1.2 kb band represents the full lengthtranscripts. The longer transcripts may be processing intermediates oralternatively spliced transcripts.

RNase H is expressed in most cell lines tested; only MRC5, a breastcancer cell line, displayed very low levels of RNase H. However, avariety of other malignant cell lines including those of bladder (T24),breast (T-47D, HS578T), lung (A549), prostate (LNCap, DU145), andmyeloid lineage (HL-60), as well as normal endothelial cells (HUVEC),expressed RNase H. Further, all normal human tissues tested expressedRNase H. Again, larger transcripts were present as well as the 1.2 kbtranscript that appears to be the mature mRNA for RNase H. Normalizationbased on G3PDH levels showed that expression was relatively consistentin all of the tissues tested.

The Southern blot analysis of EcoRI digested human and various mammalianvertebrate and yeast genomic DNAs probed with the 1.7 kb probe showsthat four EcoRI digestion products of human genomic DNA (2.4, 4.6, 6.0,8.0 Kb) hybridized with the 1.7 kb probe. The blot re-probed with a 430nucleotide probe corresponding to the C-terminal portion of the proteinshowed only one 4.6 kbp EcoRI digestion product hybridized. These dataindicate that there is only one gene copy for RNase H and that the sizeof the gene is more than 10 kb. Both the full length and the shorterprobe strongly hybridized to one EcoRI digestion product of yeastgenomic DNA (about 5 kb in size), indicating a high degree ofconservation. These probes also hybridized to the digestion product frommonkey, but none of the other tested mammalian genomic DNAs includingthe mouse which is highly homologous to the human RNase H sequence.

A recombinant human RNase H (his-tag fusion protein) polypeptide of thepresent invention was expressed in E.coli and purified by Ni-NTA agarosebeads followed by C4 reverse phase column chromatography. A 36 kDaprotein copurified with activity measured after renaturation. Thepresence of the his-tag was confirmed by Western blot analyses with ananti-penta-histidine antibody (Qiagen, Germany).

Renatured recombinant human RNase H displayed RNase H activity.Incubation of 10 ng purified renatured RNase H with RNA/DNA substratefor 2 hours resulted in cleavage of 40% of the substrate. The enzymealso cleaved RNA in an oligonucleotide/RNA duplex in which theoligonucleotide was a gapmer with a 5-deoxynucleotide gap, but at a muchslower rate than the full RNA/DNA substrate. This is consistent withobservations with E.coli RNase HI (Lima, W. F. and Crooke, S. T.,Biochemistry, 1997, 36, 390-398). It was inactive againstsingle-stranded RNA or double-stranded RNA substrates and was inhibitedby Mn²⁺. The molecular weight (˜36kDa) and inhibition by Mn²⁺ indicatethat the cloned enzyme is highly homologous to E.coli RNase HI and hasproperties consistent with those assigned to Type 2 human RNase H.

The sites of cleavage in the RNA in the full RNA/DNA substrate and thegapmer/RNA duplexes (in which the oligonucleotide gapmer had a5-deoxynucleotide gap) resulting from the recombinant enzyme weredetermined. In the full RNA/DNA duplex, the principal site of cleavagewas near the middle of the substrate, with evidence of less prominentcleavage sites 3′ to the primary cleavage site. The primary cleavagesite for the gapmer/RNA duplex was located across the nucleotideadjacent to the junction of the 2′ methoxy wing and oligodeoxynucleotide gap nearest the 3′ end of the RNA. Thus, the enzyme resultedin a major cleavage site in the center of the RNA/DNA substrate and lessprominent cleavages to the 3′ side of the major cleavage site. The shiftof its major cleavage site to the nucleotide in apposition to the DNA 2′methoxy junction of the 2′ methoxy wing at the 5′ end of the chimericoligonucleotide is consistent with the observations for E.coli RNase HI(Crooke et al. (1995) Biochem. J. 312, 599-608; Lima, W. F. and Crooke,S. T. (1997) Biochemistry 36, 390-398). The fact that the enzyme cleavesat a single site in a 5-deoxy gap duplex indicates that the enzyme has acatalytic region of similar dimensions to that of E.coli RNase HI.

Accordingly, expression of large quantities of a purified human RNase Hpolypeptide of the present invention is useful in characterizing theactivities of a mammalian form of this enzyme. In addition, thepolynucleotides and polypeptides of the present invention provide ameans for identifying agents which enhance the function of antisenseoligonucleotides in human cells and tissues.

For example, a host cell can be genetically engineered to incorporatepolynucleotides and express polypeptides of the present invention.Polynucleotides can be introduced into a host cell using any number ofwell known techniques such as infection, transduction, transfection ortransformation. The polynucleotide can be introduced alone or inconjunction with a second polynucleotide encoding a selectable marker.In a preferred embodiment, the host comprises a mammalian cell. Suchhost cells can then be used not only for production of human Type 2RNase H, but also to identify agents which increase or decrease levelsof expression or activity of human Type 2 RNase H in the cell. In theseassays, the host cell would be exposed to an agent suspected of alteringlevels of expression or activity of human Type 2 RNase in the cells. Thelevel or activity of human Type 2 RNase in the cell would then bedetermined in the presence and absence of the agent. Assays to determinelevels of protein in a cell are well known to those of skill in the artand include, but are not limited to, radioimmunoassays, competitivebinding assays, Western blot analysis and enzyme linked immunosorbentassays (ELISAs). Methods of determining increase activity of the enzyme,and in particular increased cleavage of an antisense-mRNA duplex can beperformed in accordance with the teachings of Example 5. Agentsidentified as inducers of the level or activity of this enzyme may beuseful in enhancing the efficacy of antisense oligonucleotide therapies.

The present invention also relates to prognostic assays wherein levelsof RNase in a cell type can be used in predicting the efficacy ofantisense oligonucleotide therapy in specific target cells. High levelsof RNase in a selected cell type are expected to correlate with higherefficacy as compared to lower amounts of RNase in a selected cell typewhich may result in poor cleavage of the mRNA upon binding with theantisense oligonucleotide. For example, the MRC5 breast cancer cell linedisplayed very low levels of RNase H as compared to other malignant celltypes. Accordingly, in this cell type it may be desired to use antisensecompounds which do not depend on RNase H activity for their efficacy.

Similarly, oligonucleotides can be screened to identify those which areeffective antisense agents by contacting human Type 2 RNase H with anoligonucleotide and measuring binding of the oligonucleotide to thehuman Type 2 RNase H. Methods of determining binding of two moleculesare well known in the art. For example, in one embodiment, theoligonucleotide can be radiolabeled and binding of the oligonucleotideto human Type 2 RNase H can be determined by autoradiography.Alternatively, fusion proteins of human Type 2 RNase H withglutathione-S-transferase or small peptide tags can be prepared andimmobilized to a solid phase such as beads. Labeled or unlabeledoligonucleotides to be screened for binding to this enzyme can then beincubated with the solid phase. Oligonucleotides which bind to theenzyme immobilized to the solid phase can then be identified either bydetection of bound label or by eluting specifically the boundoligonucleotide from the solid phase. Another method involves screeningof oligonucleotide libraries for binding partners. Recombinant tagged orlabeled human Type 2 RNase H is used to select oligonucleotides from thelibrary which interact with the enzyme. Sequencing of theoligonucleotides leads to identification of those oligonucleotides whichwill be more effective as antisense agents.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1

Rapid Amplification of 5′-cDNA End (5′-RACE) and 3′-cDNA End (3′-RACE)

An internet search of the XREF database in the National Center ofBiotechnology Information (NCBI) yielded a 361 base pair (bp) humanexpressed sequenced tag (EST, GenBank accession #H28861), homologous toyeast RNase H (RNH1) protein sequenced tag (EST, GenBank accession#Q04740) and its chicken homologue (accession #D26340). Three sets ofoligonucleotide primers encoding the human RNase H EST sequence weresynthesized. The sense primers were ACGCTGGCCGGGAGTCGAAATGCTTC (H1: SEQID NO: 6), CTGTTCCTGGCCCACAGAGTCGCCTTGG (H3: SEQ ID NO: 7) andGGTCTTTCTGACCTGGAATGAGTGCAGAG (H5: SEQ ID NO: 8). The antisense primerswere CTTGCCTGGTTTCGCCCTCCGATTCTTGT (H2: SEQ ID NO: 9),TTGATTTTCATGCCCTTCTGAAACTTCCG (H4; SEQ ID NO: 10) andCCTCATCCTCTATGGCAAACTTCTTAAATCTGGC (H6; SEQ ID NO: 11). The human RNaseH 3′ and 5′ cDNAs derived from the EST sequence were amplified bypolymerase chain reaction (PCR), using human liver or leukemia(lymphoblastic Molt-4) cell line Marathon ready cDNA as templates, H1 orH3/AP1 as well as H4 or H6/AP2 as primers (Clontech, Palo Alto, Calif.).The fragments were subjected to agarose gel electrophoresis andtransferred to nitrocellulose membrane (Bio-Rad, Hercules Calif.) forconfirmation by Southern blot, using ³²P-labeled H2 and H1 probes (for3′ and 5′ RACE products, respectively, in accordance with proceduresdescribed by Ausubel et al., Current Protocols in Molecular Biology,Wiley and Sons, New York, N.Y., 1988. The confirmed fragments wereexcised from the agarose gel and purified by gel extraction (Qiagen,Germany), then subcloned into Zero-blunt vector (Invitrogen, Carlsbad,Calif.) and subjected to DNA sequencing.

Example 2

Screening of the cDNA Library, DNA Sequencing and Sequence Analysis

A human liver cDNA lambda phage Uni-ZAP library (Stratagene, La Jolla,Calif.) was screened using the RACE products as specific probes. Thepositive cDNA clones were excised into the pBluescript phagemid(Stratagene, La Jolla Calif.) from lambda phage and subjected to DNAsequencing with an automatic DNA sequencer (Applied Biosystems, FosterCity, Calif.) by Retrogen Inc. (San Diego, Calif.). The overlappingsequences were aligned and combined by the assembling programs ofMacDNASIS v3.0 (Hitachi Software Engineering America, South SanFrancisco, Calif.). Protein structure and subsequence analysis wereperformed by the program of MacVector 6.0 (Oxford Molecular Group Inc.,Campbell, Calif.). A homology search was performed on the NCBI databaseby internet E-mail.

Example 3

Northern Blot and Southern Blot Analysis

Total RNA from different human cell lines (ATCC, Rockville, Md.) wasprepared and subjected to formaldehyde agarose gel electrophoresis inaccordance with procedures described by Ausubel et al., CurrentProtocols in Molecular Biology, Wiley and Sons, New York, N.Y., 1988,and transferred to nitrocellulose membrane (Bio-Rad, Hercules Calif.).Northern blot hybridization was carried out in QuickHyb buffer(Stratagene, La Jolla, Calif.) with 32P-labeled probe of full lengthRNase H cDNA clone or primer H1/H2 PCR-generated 322-base N-terminalRNase H cDNA fragment at 68° C. for 2 hours. The membranes were washedtwice with 0.1% SSC/0.1% SDS for 30 minutes and subjected toauto-radiography. Southern blot analysis was carried out in 1×pre-hybridization/hybridization buffer (BRL, Gaithersburg, Md.) with a³²P-labeled 430 bp C-terminal restriction enzyme PstI/PvuII fragment or1.7 kb full length cDNA probe at 60° C. for 18 hours. The membranes werewashed twice with 0.1% SSC/0.1% SDS at 60° C. for 30 minutes, andsubjected to autoradiography.

Example 4

Expression and Purification of the Cloned RNase Protein

The cDNA fragment coding the full RNase H protein sequence was amplifiedby PCR using 2 primers, one of which contains restriction enzyme NdeIsite adapter and six histidine (his-tag) codons and 22 bp protein Nterminal coding sequence. The fragment was cloned into expression vectorpET17b (Novagen, Madison, Wis.) and confirmed by DNA sequencing. Theplasmid was transfected into E.coli BL21(DE3) (Novagen, Madison, Wis.).The bacteria were grown in M9ZB medium at 32° C. and harvested when theOD₆₀₀ of the culture reached 0.8, in accordance with proceduresdescribed by Ausubel et al., Current Protocols in Molecular Biology,Wiley and Sons, New York, N.Y., 1988. Cells were lysed in 8M ureasolution and recombinant protein was partially purified with Ni-NTAagarose (Qiagen, Germany). Further purification was performed with C4reverse phase chromatography (Beckman, System Gold, Fullerton, Calif.)with 0.1% TFA water and 0.1% TFA acetonitrile gradient of 0% to 80% in40 minutes as described by Deutscher, M. P., Guide to ProteinPurification, Methods in Enzymology 182, Academic Press, New York, N.Y.,1990. The recombinant proteins and control samples were collected,lyophilized and subjected to 12% SDS-PAGE as described by Ausubel et al.(1988) Current Protocols in Molecular Biology, Wiley and Sons, New York,N.Y. The purified protein and control samples were resuspended in 6 Murea solution containing 20 mM Tris HCl, pH 7.4, 400 mM NaCl, 20%glycerol, 0.2 mM PMSF, 5 mM DTT, 10 μg/ml aprotinin and leupeptin, andrefolded by dialysis with decreasing urea concentration from 6 M to 0.5M as well as DTT concentration from 5 mM to 0.5 mM as described byDeutscher, M. P., Guide to Protein Purification, Methods in Enzymology182, Academic Press, New York, N.Y., 1990. The refolded proteins wereconcentrated (10 fold) by Centricon (Amicon, Danvers, Mass.) andsubjected to RNase H activity assay.

Example 5

RNase H Activity Assay

³²P-end-labeled 17-mer RNA, GGGCGCCGTCGGTGTGG (SEQ ID NO: 12) describedby Lima, W. F. and Crooke, S. T., Biochemistry, 1997 36, 390-398, wasgel-purified as described by Ausubel et al., Current Protocols inMolecular Biology, Wiley and Sons, New York, N.Y., 1988 and annealedwith a tenfold excess of its complementary 17-mer oligodeoxynucleotideor a 5-base DNA gapmer, i.e., a 17mer oligonucleotide which has acentral portion of 5 deoxynucleotides (the “gap”) flanked on both sidesby 6 2′-methoxynucleotides. Annealing was done in 10 mM Tris HCl, pH8.0, 10 mM MgCl, 50 mM KCl and 0.1 mM DTT to form one of three differentsubstrates: (a) single strand (ss) RNA probe, (b) full RNA/DNA duplexand (c) RNA/DNA gapmer duplex. Each of these substrates was incubatedwith protein samples at 37° C. for 5 minutes to 2 hours at the sameconditions used in the annealing procedure and the reactions wereterminated by adding EDTA in accordance with procedures described byLima, W. F. and Crooke, S. T., Biochemistry, 1997, 36, 390-398. Thereaction mixtures were precipitated with TCA centrifugation and thesupernatant was measured by liquid scintillation counting (BeckmanLS6000IC, Fullerton, Calif.). An aliquot of the reaction mixture wasalso subjected to denaturing (8 M urea) acrylamide gel electrophoresisin accordance with procedures described by Lima, W. F. and Crooke, S.T., Biochemistry, 1997, 36, 390-398 and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley and Sons, New York, N.Y., 1988.

12 1 286 PRT Homo sapiens 1 Met Ser Trp Leu Leu Phe Leu Ala His Arg ValAla Leu Ala Ala Leu 1 5 10 15 Pro Cys Arg Arg Gly Ser Arg Gly Phe GlyMet Phe Tyr Ala Val Arg 20 25 30 Arg Gly Arg Lys Thr Gly Val Phe Leu ThrTrp Asn Glu Cys Arg Ala 35 40 45 Gln Val Asp Arg Phe Pro Ala Ala Arg PheLys Lys Phe Ala Thr Glu 50 55 60 Asp Glu Ala Trp Ala Phe Val Arg Lys SerAla Ser Pro Glu Val Ser 65 70 75 80 Glu Gly His Glu Asn Gln His Gly GlnGlu Ser Glu Ala Lys Pro Gly 85 90 95 Lys Arg Leu Arg Glu Pro Leu Asp GlyAsp Gly His Glu Ser Ala Gln 100 105 110 Pro Tyr Ala Lys His Met Lys ProSer Val Glu Pro Ala Pro Pro Val 115 120 125 Ser Arg Asp Thr Phe Ser TyrMet Gly Asp Phe Val Val Val Tyr Thr 130 135 140 Asp Gly Cys Cys Ser SerAsn Gly Arg Arg Lys Pro Arg Ala Gly Ile 145 150 155 160 Gly Val Tyr TrpGly Pro Gly His Pro Leu Asn Val Gly Ile Arg Leu 165 170 175 Pro Gly ArgGln Thr Asn Gln Arg Ala Glu Ile His Ala Ala Cys Lys 180 185 190 Ala IleGlu Gln Ala Lys Thr Gln Asn Ile Asn Lys Leu Val Leu Tyr 195 200 205 ThrAsp Ser Met Phe Thr Ile Asn Gly Ile Thr Asn Trp Val Gln Gly 210 215 220Trp Lys Lys Asn Gly Trp Lys Thr Ser Ala Gly Lys Glu Val Ile Asn 225 230235 240 Lys Glu Asp Phe Val Ala Leu Glu Arg Leu Thr Gln Gly Met Asp Ile245 250 255 Gln Trp Met His Val Pro Gly His Ser Gly Phe Ile Gly Asn GluGlu 260 265 270 Ala Asp Arg Leu Ala Arg Glu Gly Ala Lys Gln Ser Glu Asp275 280 285 2 293 PRT Gallus sp. 2 Met Leu Arg Trp Leu Val Ala Leu LeuSer His Ser Cys Phe Val Ser 1 5 10 15 Lys Gly Gly Gly Met Phe Tyr AlaVal Arg Lys Gly Arg Gln Thr Gly 20 25 30 Val Tyr Arg Thr Trp Ala Glu CysGln Gln Gln Val Asn Arg Phe Pro 35 40 45 Ser Ala Ser Phe Lys Lys Phe AlaThr Glu Lys Glu Ala Trp Ala Phe 50 55 60 Val Gly Ala Gly Pro Pro Asp GlyGln Gln Ser Ala Pro Ala Glu Thr 65 70 75 80 His Gly Ala Ser Ala Val AlaGln Glu Asn Ala Ser His Arg Glu Glu 85 90 95 Pro Glu Thr Asp Val Leu CysCys Asn Ala Cys Lys Arg Arg Tyr Glu 100 105 110 Gln Ser Thr Asn Glu GluHis Thr Val Arg Arg Ala Lys His Asp Glu 115 120 125 Glu Gln Ser Thr ProVal Val Ser Glu Ala Lys Phe Ser Tyr Met Gly 130 135 140 Glu Phe Ala ValVal Tyr Thr Asp Gly Cys Cys Ser Gly Asn Gly Arg 145 150 155 160 Asn ArgAla Arg Ala Gly Ile Gly Val Tyr Trp Gly Pro Gly His Pro 165 170 175 LeuAsn Ile Ser Glu Arg Leu Pro Gly Arg Gln Thr Asn Gln Arg Ala 180 185 190Glu Ile His Ala Ala Cys Lys Ala Ile Glu Gln Ala Lys Ser Gln Asn 195 200205 Ile Lys Lys Leu Ile Ile Tyr Thr Asp Ser Lys Phe Thr Ile Asn Gly 210215 220 Ile Thr Ser Trp Val Glu Asn Trp Lys Thr Asn Gly Trp Arg Thr Ser225 230 235 240 Ser Gly Gly Ser Val Ile Asn Lys Glu Asp Phe Gln Lys LeuAsp Ser 245 250 255 Leu Ser Lys Gly Ile Glu Ile Gln Trp Met His Ile ProGly His Ala 260 265 270 Gly Phe Gln Gly Asn Glu Glu Ala Asp Arg Leu AlaArg Glu Gly Ala 275 280 285 Ser Lys Gln Lys Leu 290 3 348 PRTSaccharomyces sp. 3 Met Ala Arg Gln Gly Asn Phe Tyr Ala Val Arg Lys GlyArg Glu Thr 1 5 10 15 Gly Ile Tyr Asn Thr Trp Asn Glu Cys Lys Asn GlnVal Asp Gly Tyr 20 25 30 Gly Gly Ala Ile Tyr Lys Lys Phe Asn Ser Tyr GluGln Ala Lys Ser 35 40 45 Phe Leu Gly Gln Pro Asn Thr Thr Ser Asn Tyr GlySer Ser Thr His 50 55 60 Ala Gly Gly Gln Val Ser Lys Pro His Thr Thr GlnLys Arg Val His 65 70 75 80 Arg Arg Asn Arg Pro Leu His Tyr Ser Ser LeuThr Ser Ser Ser Ala 85 90 95 Cys Ser Ser Leu Ser Ser Ala Asn Thr Asn ThrPhe Tyr Ser Val Lys 100 105 110 Ser Asn Val Pro Asn Ile Glu Ser Lys IlePhe Asn Asn Trp Lys Asp 115 120 125 Cys Gln Ala Tyr Val Lys His Lys ArgGly Ile Thr Phe Lys Lys Phe 130 135 140 Glu Asp Gln Leu Ala Ala Glu AsnPhe Ile Ser Gly Met Ser Ala His 145 150 155 160 Asp Tyr Lys Leu Met AsnIle Ser Lys Glu Ser Phe Glu Ser Lys Tyr 165 170 175 Lys Leu Ser Ser AsnThr Met Tyr Asn Lys Ser Met Asn Val Tyr Cys 180 185 190 Asp Gly Ser SerPhe Gly Asn Gly Thr Ser Ser Ser Arg Ala Gly Tyr 195 200 205 Gly Ala TyrPhe Glu Gly Ala Pro Glu Glu Asn Ile Ser Glu Pro Leu 210 215 220 Leu SerGly Ala Gln Thr Asn Asn Arg Ala Glu Ile Glu Ala Val Ser 225 230 235 240Glu Ala Leu Lys Lys Ile Trp Glu Lys Leu Thr Asn Glu Lys Glu Lys 245 250255 Val Asn Tyr Gln Ile Lys Thr Asp Ser Glu Tyr Val Thr Lys Leu Leu 260265 270 Asn Asp Arg Tyr Met Thr Tyr Asp Asn Lys Lys Leu Glu Gly Leu Pro275 280 285 Asn Ser Asp Leu Ile Val Pro Leu Val Gln Arg Phe Val Lys ValLys 290 295 300 Lys Tyr Tyr Glu Leu Asn Lys Glu Cys Phe Lys Asn Asn GlyLys Phe 305 310 315 320 Gln Ile Glu Trp Val Lys Gly His Asp Gly Asp ProGly Asn Glu Met 325 330 335 Ala Asp Phe Leu Ala Lys Lys Gly Ala Ser ArgArg 340 345 4 155 PRT Escherichia coli 4 Met Leu Lys Gln Val Glu Ile PheThr Asp Gly Ser Cys Leu Gly Asn 1 5 10 15 Pro Gly Pro Gly Gly Tyr GlyAla Ile Leu Arg Tyr Arg Gly Arg Glu 20 25 30 Lys Thr Phe Ser Ala Gly TyrThr Arg Thr Thr Asn Asn Arg Met Glu 35 40 45 Leu Met Ala Ala Ile Val AlaLeu Glu Ala Leu Lys Glu His Cys Glu 50 55 60 Val Ile Leu Ser Thr Asp SerGln Tyr Val Arg Gln Gly Ile Thr Gln 65 70 75 80 Trp Ile His Asn Trp LysLys Arg Gly Trp Lys Thr Ala Asp Lys Lys 85 90 95 Pro Val Lys Asn Val AspLeu Trp Gln Arg Leu Asp Ala Ala Leu Gly 100 105 110 Gln His Gln Ile LysTrp Glu Trp Val Lys Gly His Ala Gly His Pro 115 120 125 Glu Asn Glu ArgCys Asp Glu Leu Ala Arg Ala Ala Ala Met Asn Pro 130 135 140 Thr Leu GluAsp Thr Gly Tyr Gln Val Glu Val 145 150 155 5 216 PRT Mus musculus 5 GlyIle Cys Gly Leu Gly Met Phe Tyr Ala Val Arg Arg Gly Arg Arg 1 5 10 15Pro Gly Val Phe Leu Ser Trp Ser Glu Cys Lys Ala Gln Val Asp Arg 20 25 30Phe Pro Ala Ala Arg Phe Lys Lys Phe Ala Thr Glu Asp Glu Ala Trp 35 40 45Ala Phe Val Arg Ser Ser Ser Ser Pro Asp Gly Ser Lys Gly Gln Glu 50 55 60Ser Ala His Glu Gln Lys Ser Gln Ala Lys Thr Ser Lys Arg Pro Arg 65 70 7580 Glu Pro Leu Val Val Val Tyr Thr Asp Gly Cys Cys Ser Ser Asn Gly 85 9095 Arg Lys Arg Ala Arg Ala Gly Ile Gly Val Tyr Trp Gly Pro Gly His 100105 110 Pro Leu Asn Val Arg Ile Arg Leu Pro Gly Arg Gln Thr Asn Gln Arg115 120 125 Ala Glu Ile His Ala Ala Cys Lys Ala Val Met Gln Ala Lys AlaGln 130 135 140 Asn Ile Ser Lys Leu Val Leu Tyr Thr Asp Ser Met Phe ThrIle Asn 145 150 155 160 Gly Ile Thr Asn Trp Val Gln Gly Trp Lys Lys AsnGly Trp Arg Thr 165 170 175 Ser Thr Gly Lys Asp Val Ile Asn Lys Glu AspPhe Met Glu Leu Asp 180 185 190 Glu Leu Thr Gln Gly Met Asp Ile Gln TrpMet His Ile Pro Gly His 195 200 205 Ser Gly Phe Val Gly Asn Glu Glu 210215 6 26 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 6 acgctggccg ggagtcgaaa tgcttc 26 7 28 DNA ArtificialSequence Description of Artificial SequenceSynthetic 7 ctgttcctggcccacagagt cgccttgg 28 8 29 DNA Artificial Sequence Description ofArtificial SequenceSynthetic 8 ggtctttctg acctggaatg agtgcagag 29 9 29DNA Artificial Sequence Description of Artificial SequenceSynthetic 9cttgcctggt ttcgccctcc gattcttgt 29 10 29 DNA Artificial SequenceDescription of Artificial SequenceSynthetic 10 ttgattttca tgcccttctgaaacttccg 29 11 34 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 11 cctcatcctc tatggcaaac ttcttaaatc tggc 34 12 17 DNAArtificial Sequence Description of Artificial SequenceSynthetic 12gggcgccgtc ggtgtgg 17

What is claimed is:
 1. A nucleic acid probe at least 13 nucleobases inlength, and complementary to a portion of a nucleic acid encoding ahuman Type 2 RNase H polypeptide having SEQ ID NO: 1.